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NEW HAPLOTYPES OF THE PLASMODIUM FALCIPARUM CHLOROQUINE RESISTANCE TRANSPORTER (PFCRT) GENE AMONG CHLOROQUINE-RESISTANT PARASITE ISOLATES

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  • 1 The Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia; Eijkman Institute for Molecular Biology, Jakarta, Indonesia; United States Naval Medical Research Unit No. 2, U.S. Embassy, Jakarta, Indonesia; Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea; Australian Army Malaria Institute, Brisbane, Australia

Mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene were examined to assess their associations with chloroquine resistance in clinical samples from Armopa (Papua) and Papua New Guinea. In Papua, two of the five pfcrt haplotypes found were new: SVIET from Armopa and CVIKT from an isolate in Timika. There was also a strong association (P < 0.0001) between the pfcrt 76T allele and chloroquine resistance in 50 samples. In Papua New Guinea, mutations in the pfcrt gene were observed in 15 isolates with chloroquine minimum inhibitory concentrations (MICs) of 16–64 pmol, while the remaining six isolates, which had a wild-type pfcrt gene at codon 76, had MICs of 2–8 pmol. These observations confirm that mutations at codon 76 in the pfcrt gene are present in both in vivo and in vitro cases of chloroquine resistance, and that detection of the pfcrt 76T allele could predict potential chloroquine treatment failures.

INTRODUCTION

Chloroquine has been the drug of choice for treating malaria patients for the last 50 years, but the spread of drug-resistant Plasmodium falciparum has become a major problem. In Southeast Asia, 21.9 million cases of malaria were reported in 1995 alone.1,2 Malaria is a serious problem in the eastern islands of Indonesia and in nearby Papua New Guinea. Approximately 20–30% of the population in these regions typically carry malaria parasites at any given time. In addition, 20% of consultations, 16% of hospital admissions, and 14% of hospital deaths are attributable to malaria.1,2

Papuan Indonesia (formerly Irian Jaya) and Papua New Guinea have long been plagued by drug-resistant malaria. Resistance to pyrimethamine and chloroquine in the Arso-Waris-Upper Tor River areas of Papuan Indonesia is believed to have arisen in 1959–1961 with the mass distribution of medicated chloroquine and pyrimethamine salts.3 Resistance of P. falciparum to chloroquine was reported from Kalimantan in 1973 and from Papua in 1975.4,5 An increased risk of chloroquine resistance was reported from the Jayapura region of Papua, Indonesia in the1980s.6,7 Surveys conducted during the 1990s showed high malaria prevalence rates (60–92%) and levels of chloroquine treatment failure of up to 80% among indigenous and immigrant communities of northern and central Papuan Indonesia.8–12 However, due to its safety profile, low cost, and relative success in treating mildly symptomatic malaria infections among immune and semi-immune patients, chloroquine remains the treatment of choice for malaria, and no effective alternative strategy has been developed. Molecular markers of drug resistance in P. falciparum could prove useful in defining the intensity of resistance in an individual patient and the extent and severity of the problem in communities.

The aim of this study was to examine P. falciparum chloroquine resistance transporter (pfcrt) gene haplotypes in parasite isolates with known in vivo or in vitro chloroquine resistance responses.

MATERIALS AND METHODS

The Armopa region is located on the northwestern coast of Indonesian Papua. Prior to Javanese transmigration in 1995, there were small traditional villages and a single health clinic in Armopa. No mass treatment or prophylaxis was practiced before transmigrant arrivals, and the indigenous people of Armopa did not use antimalarial drugs for prophylaxis. However, chloroquine, Fansidar® (pyrimethamine and sulfadoxine) (F. Hoffmann La Roche, Basel, Switzerland), and quinine were presumably available for treatment of clinical malaria. Transmigrants, primarily from malaria-free Java and Bali, had no previous exposure to malaria and no history of antimalarial drug use before settling in Armopa. As per national health standards, they were given chloroquine for self prophylaxis during their first three months after arrival; thereafter, treatment with only chloroquine was provided for uncomplicated cases of clinical malaria. Chloroquine is widely used for treatment of clinical malaria among transmigrants and is dispensed through a health clinic in each settlement.

Blood samples were collected in 1996–1999 from study volunteers who were immigrants to the Armopa SP1 and SP2 sites and had no history of malaria or antimalarial drug use. Patients who were positive for malaria were treated at the health center with chloroquine, Fansidar®, and quinine as the respective first-, second-, and third-line drugs for uncomplicated malaria as per the Indonesian National Health Policy. Chloroquine was given at a dose of 10 mg/kg on the first day, followed by 5 mg/kg 12, 24, and 48 hours later. A single dose of Fansidar® (1 mg/kg of pyrimethamine and 20 mg/kg of sulfadoxine) was given if there was chloroquine treatment failure. This study was carried out after obtaining informed consent from all adult participants and from parents or legal guardians of minors, and was reviewed and approved by the Ethics Committees for Protection of Human Subjects at the Ministry of Health, Republic of Indonesia, the U.S. Navy Medical Research Unit No. 2 (Jakarta, Indonesia), and The Walter and Eliza Hall Institute of Medical Research (Melbourne, Australia) and the Papua New Guinea Medical Research Advisory Committee.

RESULTS

Of 85 patients, 21 (24.7%) cases cleared their parasitemias within 72 hours, had no recurrence during 28 days of follow-up, and were classified as sensitive to chloroquine. Fifty patients had persistent or recurrent parasitemias and were classified as resistant to chloroquine. Data from 15 patient samples were excluded from analysis due to incomplete clinical histories, intercurrent infections with P. vivax, or an inability to amplify gene products. Samples were analyzed for mutations in the pfcrt gene after amplification by a polymerase chain reaction (PCR) of DNA extracted from blood samples, followed by restriction fragment length polymorphism (RFLP) analysis and DNA sequencing.13,14 The DNA from a drug-sensitive strain (D10) was used as a positive control to monitor PCR conditions. As expected, the PCR product was amplified with wild-type alleles of the pfcrt gene. No PCR products were amplified in negative controls.

The results of pfcrt mutational analysis of samples from 50 cases of chloroquine treatment failure are shown in Table 1. All 50 chloroquine-resistant samples carried the mutant pfcrt 76T allele. No mutation was detected at codon 73, but variations were found at codons 72, 74, and 75 in 50 samples: SVMNT (24), CVIET (11), CVMNT (9), and SVIET (6). Statistical analysis (chi-square test with Yates’ correction) showed that the pfcrt mutation at codon 76 was strongly associated with chloroquine resistance (P < 0.0001).15 Among the 21 chloroquine-sensitive samples, only one carried a mutated pfcrt 76 allele.

Analysis of RFLP results from amplification of chloroquine-resistant P. falciparum laboratory strains K1, W2mef, VNS, 7G8, a new isolate, 2300, from Timika on the southern coast of Indonesian Papua, and two isolates, F2382 and F1568, from Flores, Indonesia showed mutations in the pfcrt gene (Table 2). Seven of these chloroquine-resistant laboratory strains showed three different pfcrt haplotypes with mutations at codons 74, 75, and 76: CVMNT (7G8), CVIKT (2300), and CVIET (K1, W2 mef, VNS, F2382, and F1568). The wild-type haplotype CVMNK was found in the chloroquine-sensitive control strain D10.

The Wosera region of East Sepik province in Papua New Guinea is highly endemic for malaria. Mutation analysis of the genes involved in chloroquine resistance from Papua New Guinea has shown the presence of P. falciparum isolates carrying the pfcrt SVMNT haplotype, which is usually found in South American parasites, but not the CVIET haplotype of Southeast Asian isolates.16 The results of mutational analysis of 21 samples of P. falciparum obtained from malaria patients in Papua New Guinea are shown in Table 2. Fifteen of these samples with chloroquine minimum inhibitory concentrations (MICs) between 16 and 64 pmol had the 76T allele. However, six isolates with MICs of 2–8 pmol had the wild-type pfcrt allele at codons 72, 73, 74, 75, and 76. There were three pfcrt haplotypes among the 21 Papua New Guinea samples (Table 2). The wild-type haplotype CVMNK was observed in isolates with MIC values of 2–8 pmol, while the chloroquine-resistant pfcrt haplotypes CVMNT and SVMNT were observed in samples with MICs between 16 and 64 pmol.

DISCUSSION

Although chloroquine resistance in P. falciparum has been reported in Indonesia and Papua New Guinea since the early 1970s, molecular analysis and in vitro/in vivo responses to antimalaria drugs have only recently been determined in this region. This study analyzed the association of mutations in the pfcrt gene in samples of P. falciparum that had been characterized as chloroquine sensitive or resistant by in vitro or in vivo tests.14 A strong association between mutations in the pfcrt gene and chloroquine resistance (P < 0.0001) was observed in those samples from individuals who had chloroquine treatment failure in vivo or had displayed chloroquine MICs of 16–64 pmol in the in vitro test.

Studies to elucidate the molecular and biochemical mechanism of resistance to chloroquine have been in progress for more than a decade. Chloroquine resistance in P. falciparum involves decreased accumulation of the drug. However, the precise mechanism is not known.17 Mutations in the P. falciparum multidrug resistance 1 (pfmdr1) gene were implicated and involvement of at least two genes was hypothesized; mutations in both the pfcrt and the pfmdr1 genes appear to be necessary for resistance to chloroquine.13,18,19 A mutation in the pfcrt gene (located on chromosome 7) at codon 76, with a change from lysine to threonine, has been invariably found in chloroquine-resistant strains and also in chloroquine-resistant field samples from Laos, Cameroon, Mozambique, Uganda, and South America.13,20–27 Transformation of chloroquine-sensitive isolates with the Dd2 pfcrt gene sequence containing the 76T mutation consistently produced chloroquine-resistant clones, and insertion of the wild-type pfcrt gene caused resistant isolates to exhibit sensitivity to chloroquine.13 Studies on field isolates have shown the occurrence of three haplotypes of pfcrt gene alleles: CVMNK among chloroquine-sensitive isolates, CVIET among chloroquine-resistant isolates from Southeast Asia and Africa, and SVMNT among chloroquine-resistant isolates from South America and Papua New Guinea.13,16,28 The presence of the 76T pfcrt gene mutation has been correlated with risk of therapeutic failure when malaria due to P. falciparum is treated with chloroquine.22,29,30 Recently, an analysis of the genetic mutations associated with chloroquine resistance in an area highly endemic for malaria (the Wosera region of East Sepik province in Papua New Guinea) was also reported.16 All (100%) samples from treatment failures (Indonesian Papua) and 67% of the isolates (Papua New Guinea) collected prior to treatment in the in vitro studies carry the mutated pfcrt allele 76 (Tables 1 and 2).

In this study, analyses of known laboratory isolates that are resistant to chloroquine showed the presence of a mutation in the pfcrt gene. Samples collected in Papua New Guinea for in vitro chloroquine susceptibility testing provide further support for our data from clinical studies in Armopa. Although a mutated pfcrt codon 76 is invariably present in chloroquine-resistant isolates, comparison of pfcrt haplotypes revealed some interesting features. In both Armopa (Papua) and Papua New Guinea, the CVMNK haplotype was the wild type. In Papua New Guinea, the chloroquine-resistant haplotypes detected were SVMNT and CVMNT, as demonstrated in other studies (Table 3).16,28 Interestingly, the pfcrt haplotypes CVIET (African, Southeast Asian) and SVMNT (South American) were also detected in Papuan samples. In addition, two new pfcrt haplotypes were detected in Papua that have not been previously reported: SVIET, which was found in clinical samples isolated from cases of treatment failure in Armopa and CVIKT, a haplotype found in chloroquine-resistant laboratory strain 2300, which was isolated in 1985 in Timika, Papua. The presence of African, South American, Southeast Asian, and two new chloroquine-resistant haplotypes in these regions raises the question of the evolution of these five haplotypes. They may have evolved by sequential mutations of the gene in this region, where the parasite is widely circulated, or the parasites with these haplotypes were transferred into this region. This speculation on the origin of haplotypes awaits detailed studies with data from other loci in the genomes of P. falciparum isolates.

In conclusion, our results support the hypothesis that the molecular basis of chloroquine resistance involves mutations in the pfcrt gene and that detection of a mutated pfcrt allele 76 could predict potential chloroquine treatment failures.

Table 1

In vitro chloroquine responses and Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene haplotypes among malaria patients in Armopa, Indonesian Papua*

pfcrt codons
PatientSampling dateIn vivo response†7273747576
* Codon mutations are indicated in bold.
† R = resistant; S = sensitive.
‡ Sample from a patient with a recrudescent parasitemia that was not included in the statistical analysis.
CQ00412/6/96RISVMNT
CQ00812/7/96RISVMNT
CQ0103/10/97RISVMNT
CQ0104/12/97RIIISVMNT
CQ0175/2/97RIIISVMNT
CQ0391/26/97RISVMNT
CQ0394/24/97RISVMNT
CQ0419/4/97RIIISVMNT
CQ0425/8/97RIIISVMNT
CQ0444/13/97RIIISVMNT
CQ0447/8/97RIIISVMNT
CQ0545/3/97RIIISVMNT
CQ0555/4/97RIIISVMNT
CQ0605/8/97RIIISVMNT
CQ0625/14/97RIIISVMNT
CQ07212/4/96RISVMNT
CQ0914/16/97RISVMNT
CQ1011/15/97RIIISVMNT
CQ11811/9/97RIISVMNT
CQ0418/10/97RIIISVMNT
CQ04110/30/97RIIISVMNT
CQ0724/21/97RIIISVMNT
CQ0805/5/97RIIISVMNT
CQ080‡7/29/97RISVMNT
CQ07610/15/96RIIISVIET
CQ1045/2/97RIIISVIET
CQ10611/6/96RIIISVIET
CQ0203/11/97RIIISVIET
CQ1165/3/97RIIISVIET
CQ0263/15/97RIIISVIET
CQ0788/29/97RIIICVIET
CQ07811/26/97RIIICVIET
CQ0772/28/97RIIICVIET
CQ08211/8/97RIIICVIET
CQ0943/23/97RIIICVIET
CQ11310/12/97RIICVIET
CQ1182/15/97RIIICVIET
CQ1283/15/97RIIICVIET
CQ0732/3/97RIIICVIET
CQ0242/6/97RIIICVIET
CQ1312/7/97RIICVIET
CQ0031/21/97RICVMNT
CQ0593/30/97RICVMNT
CQ0674/30/97RIIICVMNT
CQ0224/4/97RIIICVMNT
CQ0224/23/97RIIICVMNT
CQ0878/1/97RIIICVMNT
CQ09512/13/96RICVMNT
CQ1054/13/97RICVMNT
CQ0364/26/97RIICVMNT
CQ0245/6/97SCVMNK
CQ0272/6/97SCVMNK
CQ0361/30/97SCVMNK
CQ0422/4/97SCVMNK
CQ0439/25/97SCVMNT
CQ0511/24/97SCVMNK
CQ0564/5/97SCVMNK
CQ0917/30/98SCVMNK
CQ0011/26/98SCVMNK
CQ0901/10/99SCVMNK
CQ0625/5/98SCVMNK
CQ07711/28/98SCVMNK
CQ0222/25/97SCVMNK
CQ0951/28/98SCVMNK
CQ05811/13/97SCVMNK
CQ02011/12/97SCVMNK
CQ06010/17/98SCVMNK
CQ0981/28/98SCVMNK
CQ01103/15/97SCVMNK
CQ01022/3/97SCVMNK
CQ1137/12/98SCVMNK
Table 2

In vitro chloroquine responses and Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene haplotypes among laboratory strains and field samples*

pfcrt codons
Strains/samplesOriginMIC507273747576
* Codon mutations are indicated in bold.
† For the laboratory strains, 50% mean inhibitory concentration (MIC50) values are in nanomoles and were determined by an in vitro microtiter assay.31 For the field isolates, values are in picomoles and were calculated at the Australian Army Malaria Institute.
Laboratory strains
    D10Papua New Guinea1CVMNK
    2300Papua75CVIKT
    K1Thailand130CVIET
    W2 mefSoutheast Asia100CVIET
    VNSVietnam80CVIET
    F2382Flores, Indonesia30CVIET
    F1568Flores, Indonesia128CVIET
    7G8South America300CVMNt
Field samples (from all Papua New Guinea)
    DR164.0SVMNT
    DR364.0SVMNT
    DR932.0SVMNT
    DR2132.0SVMNT
    DR2432.0SVMNT
    DR516.0SVMNT
    DR1216.0SVMNT
    DR1516.0SVMNT
    DR2216.0SVMNT
    DR232.0SVMNT
    DR1164.0CVMNT
    DR432.0CVMNT
    DR1864.0CVMNT
    DR2016.0CVMNT
    DR2316.0CVMNT
    DR142.0CVMNK
    DR172.0CVMNK
    DR198.0CVMNK
    DR102.0CVMNK
    DR132.0CVMNK
    DR162.0CVMNK
Table 3

Geographic distribution of Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene haplotypes among chloroquine-resistant strains of P. falciparum*

pfcrt codons
7273747576Chloroquine susceptibilityLocation
* Codon mutations are indicated in bold.
CVMNKSensitive
CVMNTResistantPapua, Papua New Guinea, South America
CVIKTResistantPapua
CVIETResistantPapua, Southeast Asia, Africa
SVIETResistantPapua
SVMNTResistantPapua, Papua New Guinea, South America

Authors’ addresses: Hadya S. Nagesha and Gerard J. Casey, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne, 3050, Australia, Telephone: 62-3-934-52479, Fax: 62-3-934-70852, E-mail: hadya@wehi.edu.au and Eijkman Institute for Molecular Biology, Jl Diponegoro 69, Jakarta 10430, Indonesia. Karl H. Rieckmann, Australian Army Malaria Institute, Brisbane, Australia. David J. Fryauff, Budi S. Laksana, Jason D. Maguire, and J. Kevin Baird, United States Naval Medical Research Unit No.2, U.S. Embassy, Jakarta, Indonesia. John C. Reeder, Papua New Guinea Instititute of Medical Research, Goroka, Papua, New Guinea.

Acknowledgments: We thank Alan Cowman for supervision and encouragement during the course of this study; Sangkot Marzuki, Syafruddin, and Graham Brown for their encouragement and support; and Jenny Thompson and Deborah Baldi for providing the D10 parasite. We also thank the Ministry of Health, Republic of Indonesia for assistance in the collection of specimens in Armopa, Papua.

Financial support: This work was supported by the Australian and Indonesian Governments through the Australian Agency for International Development and Bappenas, respectively, and the Global Emerging Infections Surveillance program of the U.S. Department of Defense. Hadya S. Nagesha was supported by a Traveling Fellowship from the Wellcome Research Trust (United Kingdom).

Disclaimer: The views of the authors expressed herein are their own and do not purport or reflect those of the U.S. Navy or the U.S. Department of Defense.

REFERENCES

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    WHO, 1987. World malaria situation 1985. World Health Stat Q 40 :142–170.

  • 2

    WHO, 1997. Malaria in the South-East Asia Region. New Delhi: Regional Office for South-East Asia, World Health Organization.

  • 3

    Meuwissen TJHE, 1964. The use of medicated salt in an antimalaria campaign in West New Guinea. Trop Geogr Med 16 :245–255.

  • 4

    Clyde DF, McCarthy VC, Miller RM, Hornick RB, 1976. Chloroquine-resistant falciparum malaria from Irian Jaya, Indonesian New Guinea. Am J Trop Med Hyg 79 :38–41.

    • Search Google Scholar
    • Export Citation
  • 5

    Ebisawa I, Fukuyama T, 1975. Chloroquine resistance of Plasmodium falciparum in West Irian and East Kalimantan. Ann Trop Med Parasitol 69 :275–282.

    • Search Google Scholar
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